PROJECT SUMMARY Alterations in chromatin structure are associated with many human diseases and often characterized by changes in histone post-translational modifications (PTMs). Histone PTMs are commonly analyzed by chromatin immunoprecipitation (ChIP), which relies on antibodies to enrich chromatin subsets. However, the accuracy of such antibodies is an increasing concern in the biomedical field, impeding advancements in chromatin science and related drugs / diagnostics. EpiCypher is pioneering the development of technologies that use recombinant designer nucleosomes (dNucs), with an early focus on PTM antibody specificity testing. Using EpiCypher?s disruptive quantitative ChIP platform (SNAP-ChIP; Sample Normalization and Antibody Profiling), we found that >80% of commercially-available PTM antibodies display a striking amount of off-target binding and low binding efficiency. These results underscore substantial problems in the selection and validation of antibodies to histone PTMs, which currently utilize histone peptides for candidate selection. Ideally, we would integrate SNAP-ChIP assays early in antibody development; however, SNAP-ChIP is low-throughput, labor intensive, and thus not suitable for antibody development. In this Direct-to-Phase II proposal we will address these deficiencies with NucleoPlex?, which couples modified dNucs to Luminex xMAP beads for multiplexed, high-throughput antibody screening. The innovation of this proposal is the creation of dNuc-xMAP bead panels (i.e. NucleoPlex panels) that enable both on- and off- target antibody profiling in a single reaction. In Phase I equivalent proof-of-concept studies, we conjugated barcoded xMAP beads to dNucs containing histone methyl-lysine PTMs, and used this panel to interrogate the binding of >50 commercially available ChIP-grade PTM antibodies. NucleoPlex data demonstrated strong concordance with SNAP-ChIP, at fraction of the time and cost, indicating that NucleoPlex provides accurate and rapid specificity screening of PTM antibodies in a nucleosomal context. EpiCypher is poised to make a significant breakthrough with the NucleoPlex platform, redefining histone PTM antibody development, screening, and validation. A major goal of this Phase II study is to integrate NucleoPlex into a recombinant antibody development pipeline, and demonstrate how this approach increases production of highly specific antibodies. To this end, we will first validate and scale manufacturing of NucleoPlex panels for distinct histone PTM families (Aim 1), and then apply NucleoPlex toward the development of recombinant antibodies (Aim 2). Finally, we will develop combinatorially-modified nucleosomes to interrogate antibody binding specificity in the context of adjacent PTMs, which have been shown to impact antibody binding (Aim 3). The development of NucleoPlex as a low-cost multiplexed antibody screening platform will accelerate the generation of highly specific histone PTM-targeting reagents, which will have a lasting impact on the epigenetics field and save millions of dollars annually that are wasted on low-quality detection reagents.